Synthesis of 2H- and 13C- substituted dithanes

ABSTRACT

The present invention is directed to labeled compounds, [2- 13 C]dithane wherein the  13 C atom is directly bonded to one or two deuterium atoms. The present invention is also directed to processes of preparing [2- 13 C]dithane wherein the  13 C atom is directly bonded to one or two deuterium atoms. The present invention is also directed to labeled compounds, e.g., [ 2 H 1-2 ,  13 C]methanol (arylthio)-, acetates wherein the  13 C atom is directly bonded to exactly one or two deuterium atoms.

STATEMENT REGARDING FEDERAL RIGHTS

[0001] This invention was made with government support under ContractNo. W-7405-ENG-36 awarded by the U.S. Department of Energy. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

[0002] The present invention relates to labeled compounds and moreparticularly to compounds labeled with carbon-13 and hydrogen-2.

BACKGROUND OF THE INVENTION

[0003] Stable isotope labeled amino acids and nucleotides are requiredfor structural and mechanistic studies of proteins and oligonucleotides.In addition, isotopically labeled biologically active compounds arerequired for many phases of drug discovery and development includingelucidation of biosynthetic pathways, pharmacokinetics, and drugmetabolism. For many applications, site-specific ¹³C or combined ¹³C and²H labeling are required. While a number of stable isotope labeledcompounds are available from companies such as Sigma-Aldrich Chemicals,a need remains for other labeled synthetic precursors.

[0004] Dithane has been used in a wide number of reactions to make alarge number of such biomolecules and other important syntheticprecursors. For example, dithane can be used as a nucleophilic synthon.While dithane could provide a chemically stable and non-volatile carrierfor the valuable ¹³C and ²H labels, the preparation of isotopicallylabeled dithane has not been previously accomplished. Thus, availabilityof ²H- and ¹³C-substituted dithanes would allow researchers to takeadvantage of the wealth of chemistry that has been done using unlabeledmethyl phenyl sulfide.

[0005] As carbon-13 is separated from its lighter isotope by cyrogenicdistillation of carbon monoxide (CO), all labeled carbons are derivedultimately from CO. The highly efficient conversion of CO to usefulchemical precursors is perhaps the most unique aspect of stable isotopelabeling technology. Any inefficiencies in the early synthetic steps addgreatly to the overall expense of isotope labeling. Thus, considerableefforts have been directed to the development of methods for thepreparation of useful synthetic precursors or synthons. This effort hasgiven rise to efficient large-scale methods for the synthesis ofmethane, methanol, methyl iodide, sodium formate, potassium cyanide andcarbon dioxide. These methods are the foundation of all labelingchemistry. The most useful of the electrophilic one-carbon precursors,methyl iodide and carbon dioxide, are difficult to store and useefficiently due to their high volatility.

[0006] As spectroscopic instrumentation and techniques continue toimprove, there is a drive to study ever more complicated bio-systems.This has lead to demands for more complex labeling patterns inbiomolecules. In the past, the simple introduction of a labeled atomsite-specifically without stereospecificity was the major thrust forstable isotope labeling and the first generation of labeled synthonsserved this effort well. Increasingly, in today's labeling climate, inaddition to site-specific labeling, the requirement forstereospecificity has been added. This includes both the ability tostereospecific label chiral compounds as well as the ability todifferentiate between prochiral centers with deuterium or carbon.Additional synthons as starting materials will address those growingdemands.

[0007] It is an object of the present invention to provide labeledcompounds.

SUMMARY OF THE INVENTION

[0008] In accordance with the purposes of the present invention, asembodied and broadly described herein, the present in invention providesthe labeled compound, [2-¹³C]dithane wherein the ¹³C is directly bondedto exactly one or two deuterium atoms.

[0009] The present invention further provides a process of preparingcompounds such as [2, 2-²H₂, 2-¹³C]dithane by reacting [²H₃, ¹³C]methylphenyl sulfoxide with sodium acetate to form an intermediate product;and, reacting said intermediate product with HS—(CH₂)₃—SH to form [2,2-²H₂, 2-¹³C]dithane.

[0010] The present invention still further provides labeled compounds,of the structure Ar—S—C*D_(2-x)H_(x)—O—R where Ar is an aryl group, C*is a ¹³C labeled carbon atom, D is a ²H, x is 0 or 1, and R is selectedfrom a C₁-C₅ lower alkyl group or an acyl group including a C₁-C₅ loweralkyl group substituent, i.e., the acyl group is (—C(O)R¹) where R¹ is aC₁-C₅ lower alkyl. In a preferred embodiment, R is an acyl group whereinR¹ is methyl such that the labeled compounds are [²H₁₋₂,¹³C]methanol(arylthio)-, acetates wherein said ¹³C atom is directlybonded to exactly one or two deuterium atoms.

DETAILED DESCRIPTION

[0011] Dithane is a useful organic reagent that allows for theconstruction of many useful biochemicals and materials. Isotopicallylabeled dithane can be used to introduce a carbon-13 and a hydrogen-2 ordeuterium label [²H] into such biochemicals and materials.

[0012] As used herein, the term “aryl” means a monovalent monocyclic orbicyclic aromatic hydrocarbon radical of 6 to 10 ring atoms, andoptionally substituted independently with one, two, three, four or fivesubstituents selected from alkyl, haloalkyl, cycloalkyl, halo, nitro,cyano, —OR (where R is hydrogen, alkyl, haloalkyl, cycloalkyl,optionally substituted phenyl), acyl, and —COOR (where R is hydrogen oralkyl). More specifically the term aryl includes, but is not limited to,phenyl, 1-naphthyl, 2-naphthyl, and derivatives thereof.

[0013] As used herein, the term “acyl” means a (—C(O)R¹ group) aryl oraliphatic acyl groups where R¹ is, e.g., a C₁₋₅ lower alkyl. Examples ofacyl include straight chain or branched alkanoyls such as formyl;acetyl, propanoyl, butanoyl, 2-methylpropanoyl, and pentanoyl.Preferably the acyl is acetyl.

[0014] In one embodiment of the invention, [2, 2-²H₂, 2-¹³C]dithane canbe made from [²H₃, ¹³C]methyl phenyl sulfoxide in process as shownbelow. The [2, 2-²H₂, 2-¹³C]dithane can be used as a non-volatilecarrier of the desired carbon and hydrogen labels.

[0015] Availability of the [2, 2-²H₂, 2-¹³C]dithane will allowresearchers to take advantage of the wealth of chemistry that has beendone using unlabeled dithane.

[0016] The present invention provides efficient large scale processesfor the preparation of [2, 2-²H₂, 2-¹³C]dithane from [²H₃, ¹³C]methylphenyl sulfoxide. Dithane provides a chemically stable and non-volatilecarrier for the valuable ¹³C and ²H labels.

[0017] In the process of the present invention, [2, 2-²H₂,2-¹³C]dithanecan be prepared in a high yield (>95%) process by first reacting [²H₃,¹³C]methyl phenyl sulfoxide with, e.g., a sodium acylate, preferablysodium acetate, in acetic anhydride to form an intermediate and reactingthe intermediate with HS—(CH₂)₃—SH in methylene chloride to produce thedithane. Optionally, the dithane may be prepared with only a singledeuterium atom on the ¹³C labeled atom by altering the deuteriumsubstitution on the [¹³C]methyl phenyl sulfoxide.

[0018] The present invention is more particularly described in thefollowing examples which are intended as illustrative only, sincenumerous modifications and variations will be apparent to those skilledin the art.

EXAMPLE 1

[0019] [¹³C]Methyl phenyl sulfide was prepared as follows. A one-liter,two-neck flask was fitted with an argon inlet adapter and an air-cooledcondenser. This flask was charged with 46.2 gram (g) (1.40 mole)[¹³C]methanol (99% ¹³C) and 726 milliliter (ml) (4.20 mole, 3.00equivalents (eq)) hydroiodic acid (HI) (47% by weight solution inwater). The air-cooled condenser was fitted with an outlet adapter,which in turn was attached (via a short piece of Tygon® tubing) to along solvent trap immersed in an ice-water bath. This ice-cooled solventtrap was connected to an inlet adapter on a two-liter, two-neck flaskcontaining a vigorously stirring biphasic mixture of 169.7 g (1.54 mole,1.10 eq) thiophenol and 140 g (3.50 mole, 2.50 eq) of sodium hydroxide(NaOH) in a mixture of 400 ml benzene and 300 ml water. The second neckof this flask was fitted with an isopropanol/dry ice-cooled condenserwith an argon outlet. The [¹³C]methanol/HI solution was then heated at85° C. for 2 hours, and then heating was discontinued. Again, any[¹³C]methyl iodide, which had collected in the ice-cooled trap wastransferred to the sodium-thiophenoxide mixture, and this mixture wasallowed to stir overnight. The mixture was then transferred to aseparatory funnel containing 400 ml of ethyl ether (Et₂O), the organicphase was washed with three 100 ml portions of water, and then driedover sodium sulfate (Na₂SO₄). Removal of the solvents under reducedpressure gave 168 g (95.6% theoretical yield) of [¹³C]methyl phenylsulfide as a clear, colorless oil: ¹H NMR (500 MHz, CDCl₃) ∂2.33-2.61(d,3H,J=139.6 Hz), 7.11-7.26(m, 5H); ¹³C NMR (125 MHz, CDCl₃) ∂ 138.4,128.8, 126.7, 125.0, 15.9.

EXAMPLE 2

[0020] [²H₃, ¹³C]Methyl phenyl sulfide was prepared as follows from[²H₄, ¹³C]methanol using the procedure of Example 1. From 36.6 g (0.987mole) of [²H₄, ¹³C]methyl alcohol, 540 ml (2.96 mole, 3.00 e q) HI (47%aqueous solution), 120 g (1.09 mole, 1.10 eq) thiophenol, and 98.7 g(2.47 mole, 2.50 eq) NaOH was obtained 125 g (98.6% theoretical yield)[²H₃, ¹³C]-methyl phenyl sulfide as a clear, slightly yellow oil: ¹H NMR(300 MHz, CDCl₃) ∂ 7.10-7.26 (m, 5H); ¹³C(75 MHz, CDCl₃) ∂ 138.4, 128.8,126.7, 125.0, {16.0, 15.7, 15.4, 15.1, 14.9, 14.6, 14.3 (septet, J=21.3Hz)}.

EXAMPLE 3

[0021] [²H₃, ¹³C]Methyl phenyl sulfoxide was prepared as follows. A 30%aqueous solution of hydrogen peroxide (19.53 g; 0.17 moles, 1.2 eq.) wasadded to a ethanol solution (184 ml) of [²H₃, ¹³C]methyl phenyl sulfide(18.4 g, 0.14 moles). The reaction was stirred at room temperature for 3days. After this period the reaction was complete and ethyl acetate (300mL) was added to the reaction. The reaction was transferred to aseparatory funnel and an equal volume of water was added. The organiclayer was recovered and the aqueous layer was extracted twice more withethyl acetate (2×300 mL). The combined organic phases are dried withNa₂SO₄ then filtered and solvents evaporated. Remaining solvent wasremoved from the solid under vacuum using a liquid nitrogen cooled trap.[²H₃, ¹³C]Methyl phenyl sulfoxide (19.45 g; 94%) was obtained as acolorless oil, pure by NMR (>98%), which could be used in subsequentreactions without further purification. ¹H [CDCl₃, 300 MHz] 7.56-7.95(m, 5H), ¹³C [CDCl₃, 75 MHz] 43.07 (heptet, J=21 Hz) 123.38, 129.26,130.94, 145.44.

EXAMPLE 4

[0022] [²H₂, ¹³C]Methanol (phenylthio)-, acetate was prepared asfollows. [²H₃, ¹³C]Methyl phenyl sulfoxide (10.0, 0.07 moles) wasdissolved in acetic anhydride (50 mL). Solid sodium acetate (NaOAc)(11.39 g, 0.14 moles) was added to the solution and the resultingmixture was heated at reflux for 24 hours. The reaction was cooled andadded to a mixture of ethyl acetate (400 mL) and saturated sodiumbicarbonate (200 mL). The biphasic mixture was stirred for 2 hours andthen separated in a separatory funnel. The aqueous layer was washed withadditional ethyl acetate (2×200 mL). The combined organic phases aredried with Na₂SO₄ then filtered and solvents evaporated. Remainingsolvent was removed from the solid under vacuum using a liquid nitrogencooled trap. [²H₂, ¹³C]Methanol (phenylthio)-, acetate (12.84 g; 90%)was obtained as a colorless oil, pure by NMR (>98%), which could be usedin subsequent reactions without further purification. ¹H [CDCl₃, 300MHz] 2.11 (s, 3H) 7.29-7.50 (m, 5H), ¹³C [CDCl₃, 75 MHz] 20.84, 67.6(pentet, J=21 Hz) 127.11, 128.92, 130.03, 134.47, 170.03.

EXAMPLE 5

[0023] [2, 2-²H₂, 2-¹³C]Dithane was prepared as follows. [²H₂,¹³C]Methanol (phenylthio)-, acetate (2 g, 0.010 moles) was dissolved intoluene (20 mL). Propane dithiol (1.40 g, 0.013 moles) was added via asyringe to the toluene solution. To this solution an equal weight ofAmberlyst® ion exchange resin (2 g) was added as a solid. The reactionmixture was refluxed for 3 days after which the reaction was complete asmonitored by NMR. The reaction was filtered and the solid Amberlyst® ionexchange resin was washed with toluene (2×50 mL). The combined organicphase was washed with brine (3×50 mL) dried over Na₂SO₄ then filteredand evaporated. The mixture, ([2, 2-²H₂, 2-¹³C]dithane and propanedithiol), was purified by column chromatography (silica gel, 10% ethylacetate, hexane). [2, 2-²H₂, 2-¹³C]Dithane (1.30 g, 99%) was obtained asa colorless oil, pure by NMR (>98%). ¹H [CDCl₃, 300 MHz] 2.11 (m, 2H)2.09-2.05 (m, 4H), ¹³C [CDCl₃, 75 MHz] 26.52, 29.52 (pentet, J=21 Hz)31.36.

[0024] Although the present invention has been described with referenceto specific details, it is not intended that such details should beregarded as limitations upon the scope of the invention, except as andto the extent that they are included in the accompanying claims.

What is claimed is:
 1. A labeled compound, [2-¹³C]dithane wherein said¹³C atom is directly bonded to exactly one or two deuterium atoms. 2.The compound of claim 1 wherein said ¹³C atom is directly bonded toexactly one deuterium atom.
 3. The compound of claim 1 wherein said ¹³Catom is directly bonded to exactly two deuterium atoms.
 4. A process ofpreparing a labeled compound, [2-¹³C]dithane wherein said ¹³C atom isdirectly bonded to exactly one or two deuterium atoms comprising:reacting [¹³C]methyl phenyl sulfoxide with sodium acylate to form anintermediate product; and, reacting said intermediate product withHS—(CH₂)₃—SH to form said labeled compound, [2-¹³C]dithane wherein said¹³C atom is directly bonded to exactly one or two deuterium atoms.
 5. Alabeled compound of the structure Ar—S—C*D_(2-x)H_(x)—O—R where Ar is anaryl group, C* is a ¹³C labeled carbon atom, D is a ²H, x is 0 or 1, andR is selected from the group consisting of C₁-C₅ lower alkyl groups andacyl groups including a C₁-C₅ lower alkyl group substituent.
 6. Thecompound of claim 5 wherein said aryl is selected from the groupconsisting of phenyl, 1-naphthyl, 2-naphthyl, and derivatives thereof.7. The compound of claim 5 wherein said aryl is phenyl.
 8. The compoundof claim 5 wherein said labeled compound is a [²H₁₋₂, ¹³C]methanol(arylthio)-, acetate.
 9. The compound of claim 8 wherein said labeledcompound is a [²H₁₋₂, ¹³C]methanol (phenlthio)-, acetate.